Motor proteins convert metabolic energy into force and displacement, generating movement in living organisms. The largest class of such proteins derives energy from the hydrolysis of ATP, and includes the myosin, dynein, and kinesin superfamilies. Despite over a century of study and the arsenal of chemical and physical approaches that has been tried, the molecular mechanism by which mechanoenzymes work remains obscure. Today, the mystery of motility is one of the outstanding problems in biology, with obvious implications in understanding the basis of motor-related disease. The advent of in vitro assays has, at last, allowed motor proteins to be studied in comparative isolation, using highly purified components interacting in defined geometries, in many cases down to the level of individual molecules. Among the motor proteins, the kinesin-microtubule system affords special advantages for study, because (1) kinesin and related proteins represent the smallest motors yet discovered, (2) processive motion can be generated by single kinesin motors, (3) the atomic structure of the kinesin motor domain bound to ADP has been solved, (4) recombinant kinesin derivatives and kinesin-related proteins can be isolated in functional form in both bacterial and eukaryotic expression systems, and (5) technology exists that can supply forces and measure displacements on the molecular scale, with high temporal and spatial resolution. Thanks, in part, to these advantages, great strides have recently been made towards establishing constraints on possible models for movement, vastly reducing the constellation of mechanisms to consider. The long-term goal of this research is to dev-elop a quantitative understanding of how kinesin proteins function, based on detailed molecular physiology combined with biochemical and biostructurai data. Specific aims include measurement of the speeds, forces, displacements, cycle timing, ATP coupling, head-head interactions, and other properties of kinesin, kinesin-related proteins, and genetically-engineered derivatives thereof. For this purpose, advanced instrumentation based on optical trapping ('optical tweezers') and optical nanometry has been developed, and will be used in experiments conducted at the single-molecule level.